Hetero-association of anticancer antibiotics in aqueous solution: NMR and molecular mechanics analysis.

In order to investigate the effect on combinations of aromatic antibiotics used in chemotherapy, the hetero-association of the antitumour antibiotics actinomycin D (AMD) with daunomycin (DAU) or novatrone (NOV) has been studied by the methods of 1D- and 2D 500 MHz 1H-NMR spectroscopy and molecular mechanics calculations. The experimental concentration and temperature dependences of the proton chemical shifts of mixtures of the aromatic drugs have been analyzed in terms of a modified statistical-thermodynamical model of hetero-association to give the equilibrium reaction constants, the thermodynamical parameters (deltaH, deltaS) of hetero-association of AMD with DAU or NOV and the limiting values of proton chemical shifts of the molecules in the hetero-complexes. The most favorable averaged structures of the 1:1 DAU-AMD and NOV-AMD hetero-association complexes have been determined using both the limiting values of proton chemical shifts of the molecules and molecular mechanics methods (X-PLOR software). The results show that intermolecular complexes between DAU-AMD and NOV-AMD are mainly stabilized by stacking interactions of the aromatic chromophores, although the DAU-AMD hetero-complex has additional stabilization, which may be explained by an intermolecular hydrogen bond between a carbonyl group of ring C of DAU and the NH group of D-Val of the pentapeptide side chain ring of AMD. The relative content of each type of molecular complex in the mixed solution has been calculated at different values of the ratio (r) of the initial concentrations of DAU and AMD. It is found that the contributions of hetero-complexes to the general equilibrium in solution are predominant at quite different values of r, viz. at r>12 for AMD with NOV and at r>2 for AMD with DAU, compared to r>0.3 for the DAU-NOV system observed previously. It is concluded that anticancer drugs have quite different affinities for formation of hetero-complexes with other aromatic antibiotics in aqueous solution, which may need to be taken into consideration for their use in combination chemotherapy.

[1H-NMR analysis of the heteroassociation of caffeine with the antibiotic actinocyl-bis(3-dimethylaminopropylamine) in aqueous solution]. / 1H-IaMP-analiz geteroassotsiatsii kofeina s antibiotikom aktinotsil-bis(3-dimetilaminopropilaminom) v vodnom rastvore.

The heteroassociation of caffeine (CAF) and the synthetic antibiotic actinocyl-bis(3-dimethylaminopropylamine) (ACT) was studied in aqueous solution by one- and two-dimensional 1H NMR spectroscopy at 500 MHz. The equilibrium reaction constants, thermodynamic parameters (delta H and delta S) of ACT heteroassociation with CAF, the limiting values of proton chemical shifts of their molecules in the heteroassociation complex, and the spatial structure of the ACT-CAF complex were determined from the experimental dependences of proton chemical shifts of the aromatic molecules on concentration and temperature. The parameters of CAF heteroassociation with the phenoxazone antibiotic actinomycin D and its synthetic analogue ACT were comparatively analyzed and conclusions were made on the crucial role of stacking interactions of the chromophores of CAF and the phenoxazone antibiotics in the formation of the heterocomplexes in aqueous solution.

[1H-NMR analysis of self association of an antibiotic actinocyl-bis(3-dimethylaminopropylamine) in water solutions]. / 1H-IaMR-analiz samoassotsiatsii antibiotika aktinotsil-bis(3-dimetilaminopropilamina) v vodnom rastvore.

The self-association of the synthetic antibiotic actinocyl-bis(3-dimethylaminopropylamine) was studied in aqueous solution by one- and two-dimensional 1H NMR spectroscopy at 500 MHz. The two-dimensional homonuclear correlation NMR techniques (TOCSY and ROESY) were used to completely assign all the proton signals of the antibiotic and to quantitatively analyze the mutual arrangement of the antibiotic molecules in their aggregates. The concentration and temperature dependences of proton chemical shifts were used to determine the equilibrium constants and the thermodynamic parameters (delta H and delta S) of the self-association, as well as the limiting values of proton chemical shifts in associates. The experimental results were analyzed using both the indefinite noncooperative and cooperative models of the molecular self-association. The calculated value of the cooperativity coefficient (sigma approximately 1.1) for our synthetic antibiotic confirmed a substantially lower anticooperative effect at the aggregation of its molecules in comparison with that of the antitumor antibiotic actinomycin D (sigma approximately 1.5). We calculated the most favorable structure of the dimeric associate of the synthetic antibiotic in aqueous solution and found that, like in the actinomycin D dimer, the antiparallel orientation of the phenoxazone chromophore planes of interacting molecules is characteristic of its dimer. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.

[Thermodynamic analysis of interaction of mitoxantrone with deoxytetranucleotide 5'-d(TpGpCpA) in the water solution based on 1H-NMR spectrophotometry]. / Termodinamicheskii analiz vzaimodeistviia antibiotika mitoksantrona s dezoksitetranukleotidom 5'-d(TpGpCpA) v vodnom rastvore po dannym 1H-IaMR-spektroskopii.

The complex formation of the antibiotic mitoxantrone (novantrone) with the deoxytetranucleotide 5'-d(TpGpCpA) in an aqueous salt solution was studied by one- and two-dimensional (2D-TOSCY and 2D-NOESY) 1H NMR spectroscopy (500 MHz). Concentration and temperature dependence of proton chemical shifts of molecules were measured. On the basis of these data, the equilibrium constants of the reaction, the relative content of various complexes as a function of concentration and temperature, the limiting values of chemical shifts of novantrone in complexes, and the thermodynamic parameters delta H and delta S of complex formation of molecules were calculated. It was concluded that the attachment sites for novantrone are pyrimidine-purine nucleotide sequences, sites d(TG) and d(CA) of the tetranucleotide duplex. The analysis of the thermodynamic parameters of the complex formation suggests that intermolecular hydrogen bonds and electrostatic interactions of the aminoalkyl chains of novantrone with the duplex d(TpGpCpA)2 play an important role in the stabilization of complexes 1:2 and 2:2. The results were compared with those obtained earlier for typical intercalators of ethidium bromide and daunomycin under identical experimental conditions.

Hetero-association of caffeine and aromatic drugs and their competitive binding with a DNA oligomer.

NMR spectroscopy has been used to elucidate the molecular basis of the action of caffeine (CAF) on the complexation with DNA of mutagens such as ethidium bromide, propidium iodide, proflavine and acridine orange, and anticancer drugs such as actinomycin D and daunomycin. The hetero-association of CAF and each of the aromatic ligands in 0.1 mol L(-1) phosphate buffer (pD=7.1) has been investigated as a function of concentration and temperature by 500 MHz 1H NMR spectroscopy and analysed in terms of a statistical-thermodynamic model, in which molecules form indefinite aggregates for both self-association and hetero-association. The analysis leads to determination of the equilibrium constants of hetero-association and to the values of the limiting chemical shifts of the heteroassociation of CAF with each of the aromatic molecules. The hetero-association constants between CAF and each of the aromatic drugs/dyes are found to be intermediate in magnitude between those for self-association of CAF and the corresponding drug/dye. The most probable structures of the 1:1 CAF + ligand hetero-association complexes have been determined from the calculated values of the induced limiting chemical shifts of the drug protons. Knowledge of the equilibrium constants for self-association of CAF and the aromatic ligands, for their hetero-association and their complexation with a DNA fragment, the deoxytetranucleotide 5'-d(TpGpCpA), enabled the relative content of each of the CAF-ligand and CAF-ligand-d(TGCA) complexes to be calculated as a function of CAF concentration in mixed solutions. It is concluded that, on addition of CAF to the solution, the decrease in binding of drug or mutagen with DNA is due both to competition for the binding sites by CAF and the aromatic molecules, and to formation of CAF-ligand hetero-association complexes in the mixed solution; the relative importance of each process depends on the drug or mutagen being considered.

[Study of the complex formation of daunomycin with deoxytetranucleotides with bases of differing sequence in an aqueous solution by 1H-NMR spectroscopy]. / Issledovanie kompleksoobrazovaniia daunomitsina s dezoksitetranukleotidami razlichnoi posledovatel'nosti osnovanii v vodnom rastvore metodom 1H-IaMR-spektroskopii.

The complex formation of the antibiotic daunomycin with deoxytetranucleotides of different base sequence in the chain, 5'-d(GpCpGpC), 5'-d(CpGpCpG), and 5'-d(TpGpCpA) in aqueous salt solution was studied by 1D and 2D (2M-TOCSY and 2M-NOESY) 1H-NMR spectroscopy. Concentration and temperature dependences of proton chemical shifts of molecules were measured. Based on these dependences, reaction equilibrium constants, relative content of various complexes depending on concentration and temperature, limiting values of chemical shifts of protons of daunomycin incorporated in various complexes, and the thermodynamic parameters delta H and delta S of complex formation were calculated. The analysis of the results enables the conclusion that the sites of predominant intercalation of daunomycin are triplet nucleotide sequences, the binding sites of the antibiotic with three consecutive GC pairs in the tetranucleotide duplex being more preferential. Daunomycin exhibits no sequence specificity upon binding to the single-stranded deoxynucleotide sequence. From the calculated values of induced chemical shifts of daunomycin protons and 2M-NOE data, the most probable spatial structures of complexes (1:2) of the antibiotic with deoxytetranucleotides were constructed. The binding of the second daunomycin molecule to both the single-stranded and duplex form of tetramers is of pronounced anticooperative mode, which is explained by the presence in the antibiotic of a positively charged amino sugar residue, which poses considerable steric constraints for the insertion of the second antibiotic molecule into the short tetranucleotide sequence. The results were compared with the data obtained under identical experimental conditions for typical intercalators proflavine and ethidium bromide.

[1H-NMR analysis of heteroassociation of caffeine with antibiotic actinomycin D in the aqueous solution]. / 1H-IaMR analiz geteroassotsiatsii kofeina s antibiotikom aktinomitsinom D v vodnom rastvore.

The molecular basis of the action of caffeine as a complex forming agent, an interceptor of aromatic drugs intercalating into DNA was studied by the example of the an anticancer antibiotic actinomycin D examined. The hetero-association of caffeine and actionomycin D was studied by one- and two-dimensional 1H-NMR spectroscopy (500 MHz). Concentration and temperature dependences of the proton chemical shifts of molecules in aqueous solution were measured. The equilibrium reaction constant of hetero-association of caffeine with actinomycin D (K = 246 +/- 48 M-1), the limiting chemical shifts of caffeine protons in complexes were determined. The most favourable structure of the 1:1 caffeine-actinomycin D hetero-complex in aqueous solution was constructed using the calculated values of the induced proton chemical shifts of molecules and the quantum-mechanical iso-shielding curves for caffeine and actinomycin D. The thermo-dynamical parameters of the hetero-complex formation between caffeine and actinomycin D were also determined. The structural and thermo-dynamical analysis showed that dispersive forces and hydrophobic interactions play the major role in hetero-association of caffeine and actinomycin D in aqueous-salt solution. The relative content of different complexes in mixed solutions containing caffeine and actinomycin D was calculated and distinctive features of the dynamic equilibrium of caffeine-actinomycin D hetero-associates were revealed as a function of concentration and temperature. It is concluded that hetero-association of caffeine and actinomycin D molecules a lowers the effective concentration of the drug in solution and hence the pharmacological activity of actinomycin D.

[1H-NMR study of complex formation between the aromatic dye ethidium bromide and the single-stranded noncomplementary deoxytetranucleotide 5'-d(CpTpGpA) in aqueous solution]. / Issledovaniie kompleksoobrazovaniia aromaticheskogo krasitelia bromistogo etidiia s odnotsepochechnym nekomplementarnym dezoksi tetranucleotidom 5'-d(CpTpGpA) v vodnom rastvore metodom 1H IaMR -spektroskopii.

Complex formation between the aromatic dye ethidium bromide (3,8-diamino-6-phenyl-5-ethylphenanthridine) and the single-stranded noncomplementary deoxytetranucleotide 5'-d(CpTpGpA) in aqueous solution was studied by one- and two-dimensional 1H NMR spectroscopy (500 and 600 MHz). Complete assignments of proton signals from the deoxytetranucleotide 5'-d(CTGA) were made using 2D-TOCSY and 2D-NOESY spectra. The concentration dependences of proton chemical shifts of the tetranucleotide were measured at T = 298 K to determine the self-association constants of tetranucleotide molecules in solution. Due to a small probability of tetranucleotide duplex formation, the complexing of dye molecules with the monomer of the tetramer plays the main role in the equilibrium of the complex in solution, which makes it possible to analyze the specificity of interactions of the aromatic ligand with the single-stranded DNA. Different schemes of complex formation were examined, and equilibrium constants for the reactions and the limiting chemical shifts of dye protons in different complexes were determined. The relative content of different types of complexes was analyzed, and specific features of the dynamic equilibrium were revealed as a function of the dye-tetranucleotide concentration ratio. The analysis indicates a sequence-specific binding of ethidium bromide to the single-stranded oligonucleotide. The structures of 1:1 complexes of the dye with the single strand of the tetranucleotide that correspond to two most probable orientations of the dye chromophore in the pyrimidine-purine T-G-site of the tetramer base sequence were constructed using the calculated values of induced chemical shifts of ethidium bromide protons.

[Study of complex formation of ethidium bromide with the self-complementary deoxytetranucleotide 5'-d(ApGpCpT) by unidimensional and two- dimensional 1H NMR spectroscopy]. / Issledovanie kompleksoobrazovaniia bromistogo étidiia s samokomplementarnym dezoksitetranukleotidom 5'-d(ApGpCpT) metodom odnomernoi i dvumernoi 1H IaMR-spektroskopii.

Complex formation of ethidium bromide (3,8-diamino-6-phenyl-5-ethyl-phenanthridine) with the self-complementary desoxytetraribonucleosidetriphosphate d(ApGpCpT) was studied in aqueous solution by uni- and two-dimensional 1H NMR spectroscopy (500 and 600 MHz). The concentration dependence of the chemical shifts of protons of the complex at a fixed temperature (T = 308 K) were measured. The schemes of complexing of the dye with the tetranucleotide which take into account eventual molecular associates in solution are discussed. Equilibrium constants of the reactions and the limiting values for the chemical shifts of the protons of ethidium bromide incorporating into the complex were calculated. Relative contents of different complex types were analyzed. The relationship between the dynamic equilibrium and dye/tetranucleotide concentration ratio was examined. The conclusion is made that, as with the complex of proflavine with the d(AGCT) examined earlier, ethidium bromide is intercalated essentially into the G-C site of the tetranucleotide being in the duplex form. The difference in intercalation between ethidium bromide and proflavine lies in the fact that ethidium bromide incorporate on the side of the narrow groove, whereas proflavine is built in on the side of the large groove. Based on the calculated values of the induced chemical shift of ethidium bromide protons and 2-d NOE spectal data, an adequate model (1:2) of the complex of the dye with tetranucleotide in solution was constructed.